Process and apparatus for the production of sterile filtered blood clotting factors

ABSTRACT

A process and apparatus for the manufacture of sterile filtered blood clotting factore from fibrinogen with an enrichment of Factor I is provided. The process includes the step of extracting blood from at least one donor, manufacturing fibrinogen from the blood extracted in a known manner, dissolving the fibrinogen in a buffer, and effecting a sterile filtration of the buffer-dissolved fibrinogen by means of a static filtration device at a pressure of less than 0.5 atmospheres.

The invention concerns a process and an apparatus for the production ofsterile filtered blood clotting factors, particularly fromcryo-precipitate and/or fibrinogen with an enrichment of the FactorsVIII and/or I.

During blood coagulation, a complicated enzymatic process converts theliquid blood into blood clots, which brings about sealing of the damagedblood vessel by the formation of a scab. Clotting difficulties arecaused by the fact that some substances necessary for clotting arelacking in the blood, or that substances which prevent clotting arepresent to a greater degree. Clotting difficulties can be successfullytreated by transfusions of blood clotting products, such as e.g.cryoprecipitate and fibrinogen with a concentrated Factor VIII or FactorI content.

The preparation of these blood clotting products can be carried out in aknown way by means of the processes described below.

According to one of the first processes, several blood donations aretaken from donors, and mixed to form a pool. The precipitation of thesolid components of the blood, namely leucocytes, thrombocytes anderythrocytes, to give a plasma for further processing, is carried out bydeposition or by centrifuging the pool. Finally, the relatively pureplasma is deep-frozen in minutes to minus 40° C. and stored at thistemperature until analytical results have been obtained. After thawingthe deep-frozen plasma to plus 2° to 4° C., cold centrifuging is carriedout at these temperatures, which precipitates a cold precipitate(cryo-precipitate) with a higher concentration of Factor VIII. Afterdissolving the cryo-precipitate in a buffer, the solution is deep-frozenin special plants and stored at about minus 40° C. When required,several of these small products can be put together and after thawing beadministered to patients with inherited or acquired clottingdifficulties.

Another process depends on the fact that cryo-precipitate dissolved in abuffer is additionally freeze-dried (lyophilised) after deep-freezing.This lyophilised cryo-precipitate can be stored in commercialrefrigerators at 2° to 8° C., and is readily available when required.The cryo-precipitate is enriched with respect to the Factors I, V, VIIIand XIII, of which the very labile Factor VIII is of the greatestimportance. When needed, the lyophilised cryo-precipitate can beredissolved in water which has been warmed to 20° to 30° C., and is thenavailable within 5 to 10 minutes.

While the two processes described above for the production of bloodclotting products are mainly used for blood donor purposes, there isanother process used by industry in which the plasma taken for thepreparation of cryo-precipitate is made from a pool of about 45 to20,000 blood donations. The plasma is, here too, subjected to a coldtreatment, centrifuged after thawing and the cryo-precipitate thusobtained in larger quantities, dissolved in a special buffer andsubjected to a final sterile filtration in a suitable plant which isrelatively large and expensive. The sterile filtered product can then bedeep-frozen and freeze dried when, as already stated, it can be storedat plus 2 to plus 8° C.

Finally, a fourth process for the preparation of cryo-precipitate withconcentrated Factor VIII content has been described, in which the plasmawhich has been deep-frozen at minus 40° C., is centrifuged in ancentrifuge at plus 20° C. for 90 minutes. During this centrifugingstage, the contents of the bag of flask are warmed to about 4° C. whilethe cryo-precipitate deposited by the cooling immediately centrifugesout to the bottom. The methods already described for the other processesregarding the storage of the cryo-precipitate obtained, also apply here.

The above methods have the following main disadvantages, which callsinto question their introduction on the grounds of efficiency, medicalconsiderations and reasons connected with the difficulty of carryingthem out.

For the first two processes, a blood plasma is used which is relativelyimpure in leucocytes and thrombocytes, so that later sterile filtrationof the cryo-precipitate obtained can lead to considerable difficultiessince the filter may become prematurely blocked. The filtration does notgive a therapeutical useful product with the filtration techniques atpresent in use.

A further disadvantage of the first process is that great technicalexpenditure is necessary for the freezing and storage, which cannot bemet by a hospital or for patients with hemophilia A, and the product canonly be transfused when needed after a longer preparation time so thatit cannot be used in cases of emergency. While this disadvantage is notpresent with the lyophilised cryo-precipitate made by the secondprocess, i.e., the technical expenditure required is small, the bloodclotting product obtained by the second process, like that obtained bythe first process, has the disadvantage that the products are notsubject to sterile filtration and contain components which are notdissolved with molecular dispersion (clear solution), but are partiallydissolved with colloidal dispersion (alum-like). Largercolloidally-dispersed particles are deposited in the capillaries andthis leads to an increased danger of micro-embolism. Withcolloidally-dispersed solutions, the end-point of the dissolutionprocess is difficult to determine because of the opacity of the product.In practice, a great deal of time is usually lost in hospitals beforethe dissolved product can be transfused. The doctor dealing with anemergency needs clotting products which can be rapidly transfused. Inaddition, it cannot be ascertained with certainty whether the product issterile or not so that the danger of bacterial infection or sepsis ontransfusion is increased.

Furthermore, the products produced by the two previous processes have nodefinite Factor VIII content.

The third process, or industrially-used process described for theproduction of cryo-precipitate from large pools, does, however, possessall the advantages of sterile filtration but with these products thereis a significantly increased danger of infection by virus hepatitis bytransfusion of infected material (Hepatitis B). It is a valid statement,that the danger of hepatitis from blood clotting products is as great asthat for whole blood in corresponding amount. E.g. the cryo-precipitatemade from 45 individual donations, carries a hepatitis risk the same as45 individual blood transfusions together. The hepatitis risk thereforeincreases with the size of the pool from which it is obtained.

The disadvantage of the fourth previously described process is that acryo-precipitate with a very high protein content is centrifuged out,and this leads to a subsequent sterile filtration which involvesblocking of the filter in the shortest possible time. Therapeuticallyvaluable sterile filtered products are therefore not obtainable usingthis process.

The invention is therefore basically aimed at the problem of creating aprocess and an apparatus of the above-mentioned type, which makes itpossible to obtain a loss-free efficient preparation of sterile,filtered, clear soluble blood-clotting products, with a highconcentration of Factor VIII or I, or a combination of these Factors,with a minimal risk for hepatitis, and so that the products are rapidlyand readily available as a result of lyophilisation.

The process according to the invention, is characterised by the factthat at least one blood extraction, which has come from a minimum numberof donors, preferably one donor, is physically separated to produceplasma, until the plasma is almost absolutely purified of cellularcomponents, the highly purified plasma is deep frozen at a lowertemperature than the temperature of about minus 22° C. which is criticalfor the preservation of Factor VIII, and is finally thawed again,preferably to about plus 2° C., the cold precipitate (cryo-precipitate)from the thawed plasma is enriched at this temperature by physicaltreatment, preferably centrifuging, and separated from the residualplasma, the concentrated cryo-precipitate is dissolved in a buffer andthen filtered under sterile conditions by means of a static filtrationusing an autoclaved filtration apparatus, at a pressure difference of<0.5 atm.

By using a single blood donation as starting material for obtaining theplasma, the risk of virus-hepatitis infection on transufsion of thecryo-precipitate obtained, is reduced to about 0.3% per single blooddonation, while this risk for obtaining cryo-precipitate from largepools in the normal way, is approximately 14% according to data given inthe technical literature.

By physically separating the plasma from the solid components of theblood, which is preferably carried out in two sequential centrifugationstages in the process according to the invention, a highly-purifiedplasma is obtained in an advantageous manner, in which almost all thesolid particles of the blood are removed. The filter cannot becomeprematurely blocked, so that the whole of the concentrate volume passesthrough the filter without loss.

Another advantageous characteristic of the process according to theinvention, is that the thawing process which follows deep-freezing, isaccelerated by the addition of heat by means of a thermostat-controlledliquid bath, and that the final temperature of the plasma supplied forcold centrifuging is about 2° C. With a higher thaw temperature and aslower thawing process, more Factor VIII in particular may go intosolution from the cryo-precipitate and no longer be centrifuged out. Italso prevents the tendency of the precipitate to entrain dissolvedproteins, and thus the protein concentration in the precipitate isrelatively small for the same Factor VIII content. The low proteincontent is necessary for the prevention of a premature blocking of thefilter.

After centrifuging, as another advantageous characteristic of theinvention, the plasma present in the cryo-precipitate layer is almostcompletely sucked off and used for the preparation of fibrinogen (Cohn-Ifraction). The cryo-precipitate obtained is dissolved in a buffer, e.g.in sodium chloride and/or sodium citrate and subjected to sterilefiltration. This sterile filtration occurs, in an advantageous manner,in a sterile chamber (laminar-flow-box) with prevention of secondarycontaminations. During the static filtration, a constant pressure of<0.5 atm is used to advantage, since otherwise the danger of gelpolarization may occur during the static filtration. This is caused bythe fact that during static filtration the direction of flow of thesolvent and the direction of the pressure is the same, so that thesolvent passes rapidly through the pores of the filter and thefibrinogens in the hydrate envelopes deposit as a gel layer in front ofthe filter and block it. This disadvantage is prevented by the so-calledovercurrent technique, in which the direction of flow of the solvent isat right angles to the pressure direction, and the fibrinogens depositedon the filter pores are broken up and a reduction in the concentrationpolarization of the particles is facilitated, but this process is notsuitable for the sterile filtration of cryo-precipitates obtained fromsingle blood donations because of the high technical expense.

After the loss-free preparation of cryoprecipitate according to theinvention, as described above, the blood clotting products after sterilefiltration are deep frozen and lyophilised.

In order to check on the fact that the sterile filtration has proceededwithout error, after each process the so-called bubble-point-test iscarried out to advantage, i.e. the filtration apparatus is subjected toa pressure, in kg/cm², which is necessary to force the pressurising gasthrough the filter wetted with water. If gas bubbles appear on thesterile filtered side of the filter at a pressure below that of thepredetermined pressure, it is an indication that the filter has beendamaged during the experiment or before the experiment.

According to the process, in addition, purification and autoclaving ofthe filtration apparatus with a newly-inserted filter combination iscarried out before any subsequent sterile filtration. In this way anyremaining hepatitis viruses or previously processed blood is preventedfrom being carried over into the next batch of blood to be processed.

The apparatus provided for carrying out the process according to theinvention, is characterized by the fact that a closed pressure vessel isused for the sterile filtration, which serves as a receiver for theblood clotting products to be subjected to sterile filtration, and whichis divided into two chambers by at least one filter layer, one of thechambers having a connection for the introduction of inert pressurisinggas, while the other chamber is connected to a discharge connection. Thepressure vessel is preferably of such dimensions as necessary for it tobe able to accept the amount of concentrate necessary for thepreparation of an effective dose of cryo-precipitate. Preferably, thepressure vessel is a polycarbonate clyinder or a stainless steelcylinder, which is clamped between two pressure flanges, these closingthe pressure vessel on the front side, and which have the appropriateconnections. The apparatus can be auto-claved in preparation for themanufacture of a blood clotting product from at least one blooddonation, so that the transfer of any residual bacteria or viruses isprevented. The auto-claving can be carried out in the normal way in anautoclave.

According to a further advantageous characteristic of the invention, thepressure vessel is divided into two chambers by a filter layer whichconsists of several filters, so that the filter used for the unfilteredproduct always has a greater pore size than the next filter. Membranefilters can be used advantageously as filters, the smallest pore sizebeing, preferably 0.22 μm. Since the individual filters are very thin,suitable spacing devices or supporting fittings can be placed betweenthem.

As already mentioned, care must be taken with membrane filters of thissize, to ensure that during static sterile filtration methods gelpolarization is prevented, since it may lead to blocking of the filterprematurely. Filters of the above-named size can be used in the processaccording to the invention, because a highly purified plasma is to beprocessed, which is almost absolutely free from cellular components,because the cryo-precipitate centrifuged out in the cold centrifuge hasa minimum protein concentration and because operation is carried outwith a pressure of <0.5 atm. so that the above-described concentrationpolarization does not occur. The whole of the cryo-precipitateconcentrate placed in the pressure vessel is therefore filtered sterilewithout loss.

For the continuous preparation of sterile filtered blood clottingproducts it is possible, according to another advantageouscharacteristic of the invention, to connect several pressure vessels inparallel and have them joined to a common pressure source, in particulara source of inert gas. Between the inert gas source and the respectivepressure vessels a pressure control and a pressure measuring apparatuscan be included. The maximum inert gas pressure for the gas source ispreferably above the bubble-point pressure of the filter with thesmallest pore size, so that the pressure control adjusts the pressurefor carrying out the sterile filtration to <0.5 atm, while for carryingout the bubble-point test after completion of the sterile filtration, itis adjusted to the bubble-point pressure selected for the correspondingfilter, which e.g. is 3.8 atm. for the above-mentioned filter with poresize 0.22 μm.

The parallel pressure vessels are preferably arranged in a laminar-flowbox, so that secondary contamination is prevented.

The removal connection on the pressure vessel is preferably connected toa detachable sterile receiver vessel. In this way it is possible toremove continuously from the sterile filtration plant, blood clottingproducts which can be used for a transfusion or for lyochilisation.

Other characteristics and advantages of the invention are indicated inthe following description of a design example by means of the diagramms.These show, as follows:

FIG. 1: a diagrammatic view of the sterile filtration apparatus forcarrying out the process according to the invention;

FIG. 2: a diagrammatic representation of equipment which is used forobtaining a concentrated cryo-precipitate with a minimum protein contentand for separating the residual plasma;

FIG. 3: a very diagrammatic representation of gel polarization during astatic filtration process;

FIG. 4: a very diagrammatic representation of gel polarization using theover current technique and

FIG. 5: a diagrammatic representation of a unit of devices connected inparallel for the continuous preparation of sterile filtered bloodclotting products.

The filtration apparatus 1, shown diagrammatically in FIG. 1, which isused for the static sterile filtration of cryo-precipitate concentrates,is built up from several parts, and consists primarily of a pressurevessel 2, which is closed on both its front sides with covers 3 and 4,which are held against the pressure vessel by means of tension screws orother similar means 5. The pressure vessel can be transparent so thatthe filtration process can be observed.

Inert gas, in particular nitrogen, may be introduced into the pressurevessel 2 through the connection 6 provided on the upper cover, while thecryo-precipitate with a small protein content and a high Factor VIIIconcentration centrifuged out by cold centrifuging, is introduces intothe pressure vessel through the connection 7.

The internal space of the pressure vessel 2, is divided into an upperand a lower chamber, 9 and 10, by a filter layer 8, the upper chamberbeing connected to the two connections 6 and 7, while the lower chamber10, is connected to a discharge connection 11.

The filter layer 8, consists of several filters, which can be held apartby spacers, the individual filters being arranged so that the filterfacing the direction of chamber 9 has the largest pore size, while thefilter adjacent to the lower chamber 10, has the smallest pore size,preferably 0.22 μm. Membrane filters are used as filters.

In FIG. 2 are shown, diagrammatically, two containers, namely acontainer 12 in which there is the cryo-precipitate 13, centrifuged outduring cold centrifuging, and the residual plasma 14, which is directlysucked out from above the cryo-precipitate layer by means of a hollowneedle 15, a flexible tube 16 to a low pressure source 17, or a suctionpump connected to the flexible tube 16, and then collected in thecontainer 18. The plasma in the collection container, is used again forthe preparation of fibrinogen.

The cryo-precipitate collected in the container 12 has, according to theinvention, a high factor VIII concentration, a very low proteinconcentration and virtually no blood particles.

In the static filtration process shown very diagrammatically in FIG. 3,the direction of flow, indicated by the arrow 19, and the pressuredirection, indicated by the arrow 20, are the same at right angles tothe filter 21. If too high a pressure is selected for the static sterilefiltration, then the solvent, 22, passes too rapidly through the pores23, so that fibrinogen 25, enclosed in a hydrate envelope 24, isdeposited as a layer on the filter. The filter is thus blocked up in avery short time, so that not all the quantity of cryo-precipitatedissolved in the buffer, is sterile filtered and consequently the lossin efficiency is considerable.

In order to achieve the projected loss-free sterile filtration, it isnecessary to prevent gel polarization by using a low pressuredifferential, to use a highly purified blood plasma and to prepare thecryo-precipitate concentrate with a minimum protein content.

FIG. 4 shows the well-known overcurrent technique, which cannot be usedfor the process according to the invention because of the high technicalexpense. With this technique, the direction of flow 19, is at rightangles to the pressure direction 20, so that the fibrinogens do notcollect so quickly on the pores 23 of filter 21 and block them up.

Finally, FIG. 5 shows a unit of pieces of apparatus 1, connected inparallel, of which only one apparatus is shown diagrammatically forsimplicity. The individual pieces of apparatus are connected to apressure gas header tube 26, which is connected to a source of inert gas27. Each filtration apparatus 1, is connected to the pressure gas headertube 26 by means of a manometer 28 and a pressure control 29 on a branchline 30.

In order to carry out the sterile filtration with the filtrationapparatus 1, which has been previously autoclaved with the filter layer8 already inserted, the cryo-precipitate dissolved in buffer isintroduced into the pressure vessel 2, through the connection 7. Then apressure of less than 0.5 atm is applied by means of the pressurecontrol 29, and the filtration begins. Of course, the filtration processcan be carried out with the filtration apparatus beside one another, orin sequence, so that the sterile filtered blood clotting productscollected in the sterile receiver 31 can be removed continuously.

After completion of a filtration process, the so called bubble-pointtest is carried out, in which the bubble-point pressure predeterminedfor the filter used is set. It is necessary that the maximum pressure ofthe inert gas source 27, is greater than the bubble-point pressure.

The filtration apparatuses connected in parallel, are in a laminar flowbox 32, i.e., in a sterile chamber, into which purified air flows fromoutside through the sterile filter 33, in the direction of arrow 34.

In accordance with a preferred embodiment of the invention, sterilefiltered blood clotting products may be produced from fibrinogen with anenrighment of Factor 1. Initially, as before, blood is extracted from atleast one (and preferably a single) donor and the blood extracted isphysically separated (preferably by centrifuging) to obtainsubstantially purified plasma, free of cellular components. Then, thepurified plasma is deep frozen at a temperature below about minus 22° C.Subsequently, the plasma is thawed to produce a cryo-precipitate. Then,all of the plasma present above the cryo-precipitate layer is drawn offby suction to permit manufacture of fibrinogen in a known manner. Inparticular, this plasma is cooled down to 0° C. and mixed with alcoholso that a solution arises, containing 8% alcohol. Alcohol is added whilestirring the mixture constantly so that no zones appear where thealcohol content is higher than 8%. The temperature of the mixing processis constantly monitored so that it remains always at minus 1°-2° C. Atthe end of the mixing process a precipitate of fibrinogen appears whichis separated from the rest of the mixture by centrifugation. Finally,this fibrinogen precipitate is dissolved in a buffer and is then sterilefiltered in the manner previously described to produce a sterilefiltered blood clotting factor with a high concentration of Factor 1.

While only several embodiments of the present invention have been shownand described, it will be obvious to those persons of ordinary skill inthe art, that many changes and modifications may be made thereunto,without departing from the spirit and scope of the invention.

What is claimed is:
 1. A process for the manufacture of sterile filteredblood clotting products from fibrinogen with an enrichment of Factor 1,comprising the steps of:extracting blood from at least one donor;physically separating the blood to obtain substantially purified plasma,free of cellular componets, said separating step being carried out in asequence of at least two centrifuging steps, to obtain plasma with anincreasing degree of purity; deep freezing the purified plasma at atemperature below about minus 22° C.; thawing the plasma to produce acyro-precipitate; drawing off the plasma present above thecyro-precipitate and manufacturing fibrinogen from said plasma in aknown manner; dissolving the fibrinogen in a buffer; and effecting asterile filtration of the buffer-dissolved fibrinogen by means of astatic filtration device at a pressure of less tha 0.5 atm.
 2. Theprocess according to claim 1, wherein said blood is extracted from asingle donor.
 3. The process according to claim 1, wherein said sterilefiltration step is carried out in a sterile chamber to prevent secondarycontamination.
 4. The process according to claim 1, additionallyincluding the step of carrying out a bubble-point test after saidsterile filtration step, to ensure that the process has proceededwithout error.
 5. The process according to claim 1, wherein said processsteps are repeated following purification and autoclaving of thefiltration apparatus employed.
 6. The process according to claim 1,wherein said manufacturing step comprises the following substeps:coolingthe plasma drawn off down to 0° C. and mixing said cooled plasma withalcohol so as to achieve a solution containing about 8% alcohol and tothereby obtain a fibrinogen precipitate; and physicially separating saidfibrinogen precipitate from said solution by centrifuging.